ABL Kinases
The ABL family of non-receptor tyrosine kinases, ABL1 (also known as cABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration, and invasion (Bradley et al., J. Cell Sci., 2009; Colicelli, Sci. Signal., 2010; Pendergast, Adv. Cancer Res., 2002). ABL1 was first discovered as the oncogene in the Abelson murine leukemia virus (v-ABL) and was subsequently identified as an oncogene associated with chromosome translocations in BCR-ABL1-positive human leukemias. ABL tyrosine kinases play an oncogenic role in human leukemias (Wong, et al., Ann. Rev. Immunol., 2004; Greuber et al., Nat. Rev. Cancer, 2013) and promote the progression of solid tumors (Greuber et al., Nat. Rev. Cancer, 2013; Ganguly et al., Genes Cancer, 2012). ABL kinases elicit pro-tumorigenic or anti-tumorigenic effects in breast cancer cells and promote cancer cell invasion (Blanchard et al., PLOS One, 2014; Gil-Henn et al., Oncogene, 2012; Sirvent et al., Oncogene, 2007; Srinivasan et al., Oncogene, 2008).
Pioneering studies on the ABL tyrosine kinases opened the door to seminal discoveries of the molecular basis of cancer. Among these was the finding that structural alterations of the cellular ABL (c-ABL, ABLI) tyrosine kinase as a consequence of viral fusion (Gag-ABL) and chromosomal translocation (BCR-ABL1) events promote leukemia in mice and humans, respectively (Wong et al., Ann Rev. Immunology, 2004). The Gag-ABL and BCR-ABL1 fusion proteins are constitutively active and drive cellular transformation. By contrast, the kinase activities of ABL1 and ABL2 are tightly regulated by intra- and intermolecular interactions as well as by phosphorylation (Colicelli et al., Sci. Signal., 2010; Panjarian et al., J. Biol. Chem., 2013).
Studies of the cell of origin of BCR-ABL-positive chronic myeloid leukemia (CML) demonstrated its presence in hematopoietic stem cells (HSCs). The recognition that small-molecule tyrosine kinase inhibitors (TKIs) could effectively treat human CML ushered in the era of targeted cancer therapies (Eide et al., Curr. Hem. Malignancy Reports, 2015). Subsequently, the emergence of resistance to ATP-competitive inhibitors of the BCR-ABL1 kinase led to the identification of diverse drug resistance mechanisms and provided a road-map for the development of alternative therapies in the treatment of leukemias and other malignancies.
ABL Structural Domains and Enzymatic Regulation
ABL1 and ABL2 share N-terminal regulatory and catalytic domains that are over 90% identical and include the Src homology 3 (SH3), SH2, and SH1 (tyrosine kinase) domains (FIG. 1). The C-terminus of both ABL kinases contains a conserved filamentous (F) actin-binding domain. ABL1 contains a G-actin (globular actin)-binding domain upstream of the F-actin-binding domain, whereas ABL2 has a second internal F-actin-binding domain and a microtubule-binding domain which are not found in ABL1 (FIG. 1). The ABL kinases share conserved PXXP motifs that mediate binding to SH3 domain-containing proteins. ABL1 has three nuclear localization signal (NLS) motifs and one nuclear export signal (NES) in its C-terminus, which mediates its nuclear-cytoplasmic shuttling.
By contrast, ABL2, which lacks the NLS motifs, localizes primarily to the cytoplasm and preferentially accumulates at F-actin-rich sites in the cell periphery, focal adhesions, adherens junctions, invadopodia, and phagocytic cups (Bradley et al., J. Cell Sci., 2009). Alternative splicing of the first exons produces various ABL1 and ABL2 isoforms with distinct N-terminal sequences (FIG. 1). The 1b isoforms of both ABL kinases contain an N-terminal glycine that is myristoylated, while the 1a variants lack this site and the corresponding modification.
Multiple intramolecular interactions mediate ABL auto-inhibition and include the binding of the SH3 domain to the polyproline-containing linker sequence connecting the SH2 and kinase domains, as well as interactions of the SH2 domain with the C-terminal lobe of the kinase domain (SH1), leading to the formation of a SH3-SH2-SH1 clamp structure (Hantschel et al., Nature Rev. Mol. Cell Bio., 2004). The auto-inhibited conformation of ABL kinases is stabilized by the binding of the myristoylated residue in the ABL N-terminus to a hydrophobic pocket within the C-lobe of the kinase domain in the myristoylated 1b isoform of the ABL kinases (FIG. 1). In addition, intermolecular interactions with distinct binding partners can negatively or positively regulate ABL kinase activity (Colicelli et al., Sci. Signal., 2010). Intermolecular interactions that disrupt auto-inhibitory interactions result in stabilization of the active conformation of the ABL kinases and increased enzymatic activity. By contrast, intermolecular interactions that stabilize the inactive conformation of the ABL kinases inhibit enzymatic activity and downstream signaling. The activity of the ABL kinases can also be modulated by interactions with lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), which inhibits the ABL kinases in vitro and in cells; experimentally decreasing cellular PIP2 levels stimulates ABL kinase activity (Plattner et al., Nat. Cell Biol., 2003).
The enzymatic activity of the ABL kinases can also be regulated by tyrosine phosphorylation (Colicelli et al., Sci. Signal., 2010). This modification occurs in trans for both ABL1 and ABL2, and it is referred to as ‘auto-phosphorylation.’ ABL family kinases are also phosphorylated by SRC family kinases and receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). Phosphorylation of key residues in the activation loop located at the interface between the small and large lobes of the catalytic domain of protein tyrosine kinases is necessary to achieve high catalytic activity. Among the tyrosine residues phosphorylated on ABL1 are Y412 in the activation loop (corresponds to ABL2 Y439) and Y245 in the SH2-kinase domain linker (corresponds to ABL2 Y272) (FIG. 1). Phosphorylation of these sites stabilizes the active ABL conformation, leading to enhanced signaling.
The presence of common and unique domains in ABL1 and ABL2 suggests that these kinases may exhibit overlapping as well as unique functions. The unique domains present in ABL1 and ABL2 control their differential subcellular localization and/or association with distinct protein complexes, leading to diverse functional roles for these kinases in various cell types.
Physiological Roles of Murine ABL Kinases
ABL1 and ABL2 function redundantly in some cellular contexts, but also have unique roles during mouse development and physiology in the adult. Analysis of mice with tissue-specific deletion of ABL1 and/or ABL2 revealed dependence of role on the cell type (Greuber et al., J. Immunology, 2012; Gu et al., J. Immunology, 2007; Chislock et al., Proc. Natl. Acad. Sci. USA, 2013; Wetzel et al., Mol. Cell. Biol., 2012). Consistent with redundant roles for the murine ABL kinases, mice with global inactivation of both ABL1 and ABL2 die before embryonic day 11 (Koleske et al., Neuron, 1998). ABL1 single-knockout mice are viable or exhibit perinatal lethality, depending on the strain, and display phenotypes distinct from those presented by ABL2 global knockout mice (Schwartzberg et al., Cell, 1991; Tybulewicz et al., Cell, 1991; Qiu et al., Proc. Natl. Acad. Sci. USA, 2010; Moresco et al., J. Neurosci., 2005; Li et al., Nature Genetics, 2000; Kua et al., Nat. Cell Biol., 2012; Gourley et al., Proc. Natl. Acad. Sci. USA, 2009). Disruption of murine ABL1 on a mixed (129/SvEv and C57BL/6J) genetic background resulted in neonatal lethality in about 50% of the mice, a phenotype that was more severe on the C57BL/6J genetic background (Schwartzberg et al., Cell, 1991; Tybulewicz et al., Cell 1991). ABL1 knockout mice exhibit splenic and thymic atrophy, reduced numbers of B- and T-cells, cardiac abnormalities, and osteoporosis linked to defective osteoblast proliferation and premature senescence (Schwartzberg et al., Cell, 1991; Tybulewicz et al., Cell, 1991; Qiu et al., Proc. Natl. Acad. Sci. USA, 2010; Li et al., Nature Genetics, 2000; Kua et al., Nat. Cell Biol., 2012). By contrast, ABL2 (Arg) knockout mice are viable and exhibit neuronal defects that include age-related dendrite destabilization and regression (Koleske et al., Neuron, 1998; Moresco et al., J. Neurosci., 2005; Gourley et al., Proc. Natl. Acad. Sci. USA, 2009; Zheng et al., Cancer Cell, 2014). Conditional knockout mice with tissue-specific deletion of the ABL kinases revealed unique and overlapping roles for these kinases in neuronal cells, immune cells (T cells and myeloid cells), smooth muscle cells, and in cardiovascular development and function (Koleske et al., Neuron, 1998; Greuber et al., J. Immunology, 2012; Chislock et al., Proc. Natl. Acad. Sci. USA, 2013; Wetzel et al., Mol. Cell. Bio., 2012; Gu et al., Sci. Signal. 2012; Cleary et al., Resp. Res., 2013). For example, ABL kinases have redundant roles in mature T cells because a deletion of both ABL1 and ABL2 was necessary to inhibit TCR induced proliferation and cytokine production, as well as chemokine-induced migration (Gu et al., J. Immunol., 2007; Gu et al., Sci. Signal., 2012; Trampont et al., Mol. Cell. Bio., 2015). ABL1 has a unique role in airway smooth muscle because disruption of ABL1 in these cells attenuated airway hyper-responsiveness and remodeling in a mouse model of allergen-induced asthma (Cleary et al., Resp. Res., 2013). Distinct cellular functions of ABL1 and ABL2 might be mediated by their unique domains, differential subcellular localization, and/or association with distinct protein complexes.
ABL Activation in Leukemias and Development of Targeted Therapies
Chromosomal translocations are the hallmark of oncogenic activation of the ABL kinases in human leukemias (Ren et al., Nat. Rev. Cancer, 2005). Disruption of inhibitory ABL1 intramolecular interactions in Philadelphia positive (Ph+) human leukemias occurs as a consequence of the t(9; 22)(q34; q11) chromosome translocation that generates BCR-ABL1 fusion proteins with constitutive tyrosine kinase activity (FIG. 1). Chronic myelogenous leukemia (CML) begins with a chronic phase (CP-CML) that is characterized by expansion of the myeloid lineage and retention of hematopoietic differentiation (Wong et al., Ann. Rev. Immun., 2004). This early phase can progress to a blastic phase (BP-CML) characterized by reduced cellular differentiation and displacement of mature cells with immature blasts. The majority of BP-CML patients harbor several genetic alterations in addition to BCR-ABL1. Three different BCR-ABL1 proteins have been identified that differ in the amount of BCR sequences retained in the fusion protein, leading to distinct types of leukemia: P210 BCR-ABL1 is causal in chronic myelogenous leukemia (CML); P185 BCR-ABL1 is found in 20-30% of adult and 3-5% of childhood B-cell acute lymphocytic leukemia (B-ALL); and P230 BCR-ABL1 is associated with neutrophilic CML and rare cases of CML (Advani et al., Leukemia Res, 2002). Oncogenic activation of ABL1 in the BCR-ABL1 fusion protein is dependent on the presence of the BCR N-terminal coiled-coil (CC) oligomerization domain. Multiple signaling pathways have been identified that function to mediate the oncogenic activity of BCR-ABL1, and include the RAS, NF-kB, PI3K/AKT, JUN, b-catenin, and STAT signaling pathways (Ren et al., Nat. Rev. Cancer, 2005).
Oncogenic activation of the ABL kinases via chromosomal translocations has also been shown to occur in Ph-negative human leukemias (Roberts et al., N. Engl. J. Med., 2014; Kawai et al., Leukemia Res., 2014; De Braekeleer et al., Eur. J. Haem., 2011). ABL1 has been identified as a fusion partner with a number of genes in T cell acute lymphoblastic leukemia (T-ALL), B-ALL, AML, and other leukemias (FIG. 1). The ABL kinase fusions identified in a precursor B-ALL subtype lacking the BCR-ABL1 fusion (designated Ph-like ALL) are associated with poor outcome among children and adolescents (Roberts et al., N. Engl. J. Med., 2014). Similarly to BCR-ABL1, several translocations retain the ABL1 SH3 and SH2 domains. Among these are the N-terminal fusion partners: ETV6 (TEL), EML1, NUP214, ZMIZ1, and SEPT9 (Roberts et al., N. Engl. J. Med., 2014; Kawai et al., Leukemia Res., 2014; De Braekeleer et al., Eur. J. Haem., 2011). Other translocations fuse N-terminal sequences present in RCSD1, SFPQ, FOXP1, and SNX2 to the ABL1 SH2 domain and lack the SH3 domain (FIG. 1). Chimeric fusions involving the ABL2 gene have also been identified in rare leukemias. ETV6 and ZC3HAV1 are fused to ABL2 sequences upstream of the SH3 and SH2 domains, while RCSD1 and PAG1 are fused to the ABL2 SH2 domain (FIG. 1). Some fusion partners encode proteins that contain coiled-coil or helix-loop-helix motifs that promote oligomerization of the resulting chimeric proteins, leading to enhanced ABL kinase activity. However, the NUP-ABL1 fusion requires localization to the nuclear pore complex rather than oligomerization for enhanced transforming activity (De Keersmaecker et al., Mol. Cell, 2008).
Therapeutic Activity of ABL Kinases
The most successful example of molecular targeted therapy to date has been the development of tyrosine kinase inhibitors (TKIs) against BCR-ABL1 for the treatment of CML in the chronic phase (Table 1).
TABLE 1Selective and Non-selective ABL Kinase InhibitorsAlternativeInhibitorRegulatoryYear ofNameNameTargetsTypeStatusApprovalCompanyImatinibGleevec/STI571ABL1, ABL2,ATP-site,FDA2001NovartisBCR-ABL1,Type IIapproved forCSF1R, DDR1,CML, Ph+KIT, NQO2,ALL,PDGFR1MDS/MPD,ASM,HES/CEL,DFSP, GISTDasatinibSprycel/BMS-ABL1, ABL2,ATP-FDA2006Bristol-354825BCR-ABL1,competitive,approved forMyersBLK, BTK,Type ICML, Ph+SquibbCSK, CSR1R,ALLCompanyDDR1, DDR2,EGFR, ERBB2,FGR, FRK,FYN, GAK,GCK, HCK,ILK, KIT, LCK,LIMK1,LIMK2, LYN,MAP2K,MAP2K,MAP4K,PDGFR, RIPK2,SLK, SRC,SYK, TEC,TYK2, YES1NilotinibTasigna/AMN107ABL1, ABL2,ATP-site,FDA2007NovartisBCR-ABL1,Type IIapproved forCSF1R, DDR1,CMLDDR2, KIT,NQO2, PDGFRBosutimbBosulif/SKI-ABL1, ABL2,ATP-FDA2012Pfizer Inc.606BCR-ABL1,competitive,approved forCAMK2G,Type ICMLCDK2, HCK,LYN,MAPKK1,MAPKK2,MAPKKK2,SRCPonatinibIclusing/AP24534ABL1, ABL2,ATP-site,FDA2012AriadBCR-ABL1,Type IIapproved forPharmaceuticalsBLK, CSFR1,CML, Ph+Inc.DDR1, DDR2,ALLEPHRs, FGFR1,FGFR2, FGR,FLT2, FRK,FYN, HCK,LCK, LYN,RET, SRC,TEK, TIE2,TRKA, TRKB,TRKC, PDGFR,VEGFR1,VEGFR2,VEGFR3, YES1AxitinibInlyta/AG013736BCR-ABL1ATP-FDA2012Pfizer Inc.(T3151), KIT,competitive,approved forPDGFR,Type IRenal CellVEGFR1,CarcinomaVEGFR2,VEGFR3VandetanibCaprelsa/ZD-ABL1, EGFR,ATP-site,Thyroid2011AstraZeneca6474RET, VEGFRType IICancerGNF2,ABL1, ABL2,AllostericNot FDANovartisGNF5BCR-ABL1approvedABL001ABL1, ABL2,AllostericPhase I TrialNovartisBCR-ABL1for CMLand Ph+ALLThe majority of CP-CML patients treated with the BCR-ABL1 inhibitor imatinib (Gleevec; STI571) as first-line therapy have durable remissions with five-year overall and progression free-survival rates approaching 90% (O'Hare et al., Nat. Rev. Cancer, 2012). However, imatinib is less effective for the treatment of blast crisis CML and Ph+ B-ALL patients. Several second- and third-generation TKIs targeting BCR-ABL1 have been approved or are under development for CML patients who are resistant or intolerant to imatinib (Table 1). Among these are dasatinib and nilotinib, which have been FDA- and European Medicines Agency (EMA)-approved as both frontline and second-line therapies, and bosutinib and ponatinib which have been FDA- and EMA-approved for second-line therapy to treat Ph+ leukemia patients with BCR-ABL1 kinase domain mutations (Eide et al., Curr. Hem. Malignancy Reports, 2015). Recently, axitinib, a vascular endothelial growth factor receptor (VEGFR) kinase inhibitor approved for second-line therapy of refractory renal cell carcinoma, was reported to potently inhibit the BCR-ABL1 (T315I) gatekeeper mutation, which confers resistance to imatinib, dasatinib and nilotinib (Pemovska et al., Nature, 2015). Threonine (T) 315 is known as the gatekeeper residue because it is found at the periphery of the nucleotide-binding site of the ABL1 kinase within the hinge region of the enzymatic cleft (Nagar et al., Cancer Res., 2002). T315 stabilizes the binding of imatinib, dasatinib and nilotinib through a hydrophobic pocket in the active site, and thus the T315I mutation elicits complete insensitivity to these ATP-competitive inhibitors. Interestingly, axitinib preferentially inhibits the BCR-ABL1 (T315I) mutant over wild-type BCR-ABL1 (Pemovska et al., Nature 2015). Thus, axitinib might be useful for the treatment of BCR-ABL1 (T315I)-driven CML and Ph+ B-ALL. Ponatinib also inhibits the BCR-ABL1 (T315I) mutant. The effectiveness of the ABL TKIs for the treatment of Ph-negative leukemias associated with multiple ABL fusion partners remains to be established.
ABL TKIs can be classified into three main classes based on their mechanism of action. The ATP-competitive inhibitors can be sub-classified into type 1 inhibitors targeting the active conformation of the kinase domain (dasatinib, bosutinib), and type 2 inhibitors targeting the inactive conformation of the kinase domain (imatinib, nilotinib, ponatinib). The third main class includes the allosteric inhibitors which do not target the ATP-binding pocket, but instead bind to regulatory domains to inhibit kinase activity.
Notably, the ATP-competitive kinase inhibitors imatinib, dasatinib, nilotinib, bosutinib, and ponatinib have broad target specificity and inhibit multiple tyrosine kinases in addition to ABL kinases (Table 1). Axitinib has a more restricted target specificity compared to other FDA-approved ATP-competitive inhibitors because it only targets KIT, PDGFRα, and VEGFRs in addition to the BCR-ABL1 (T315I) mutant kinase.
Among allosteric TKIs targeting ABL are GNF2 and GNF5, which bind to the myristoyl-binding pocket in the C-lobe of the ABL kinase domain (Table 1) (Zhang et al., Nature, 2010). In contrast to ATP-competitive inhibitors that target multiple kinases, the allosteric inhibitors are highly selective for the ABL kinases. These allosteric inhibitors were shown to inhibit BCR-ABL1-driven leukemogenesis in mice and sensitize mutant BCR-ABL1 to inhibition by ATP-competitive TKIs (Zhang et al., Nature, 2010). A Phase I, multicenter clinical trial with a novel allosteric inhibitor of BCR-ABL1 (ABL001; U.S. Patent App. Pub. No. 2013/0310395) that targets the myristoyl-binding pocket is currently ongoing for patients with refractory CML or Ph+ B-ALL (http://clinicaltrials.gov/show/NCT02081378) (Table 1).
Several studies have reported inhibitory and, in some cases, stimulatory effects of imatinib, nilotinib, dasatinib, and other TKIs on cancer cell proliferation, survival, and motility (Ganguly et al., Oncogene, 2012; Matei et al., Clin. Cancer Res., 2004; Stahtea et al., Int. J. Cancer, 2007). However, the cellular responses to these compounds cannot be solely attributed to inhibition of the ABL kinases because these compounds target numerous kinases and some non-kinase enzymes. Furthermore, TKIs such as nilotinib, imatinib and dasatinib were shown to have off-target effects leading to the formation of BRAF/RAF1 dimers and ERK activation in several cancer cell types (Packer et al., Cancer Cell, 2011). By contrast, paradoxical activation of RAF-ERK signaling was not induced by treatment of these cancer cells with allosteric inhibitors targeting the unique myristate-binding site in the ABL kinase domain.
ABL Kinases in Solid Tumors
Recently, the ABL family kinases, ABL1 and ABL2 have been shown to play a role in the progression of several solid tumors through activation mechanisms distinct from those involved in the generation of ABL-induced leukemias. Preclinical studies on small-molecule inhibitors of the ABL kinases suggest that their use may be of benefit in the treatment of selected solid tumors.
Activation of ABL kinases in solid tumors is not linked to chromosome translocation events as found in human leukemias, but instead is driven by enhanced ABL1 or ABL2 expression and/or activation due to amplification, increased gene expression, enhanced protein expression, and/or increased enzymatic activity in response to stimulation by oncogenic tyrosine kinases, chemokine receptors, oxidative stress, metabolic stress, and/or inactivation of negative regulatory proteins (Lin et al., Oncogene, 2008; Ganguly et al., Oncogene, 2012; Nature, 2012; Cerami et al., Cancer Discov., 2012; Sos et al., J. Clin. Invest., 2009; Simpson et al., J. Urol., 2005; Behbahani et al., World J. Urology, 2012).
The Cancer Genome Atlas (TCGA) and other large-scale sequencing projects report ABL amplification, somatic mutations, and/or increased mRNA expression in multiple solid tumors (www.cbioportal.org). These genomic alterations are more common in ABL2 than ABL1, with ABL2 alterations being observed in 24% of liver hepatocellular carcinomas, and to a lesser extent in uterine endometrioid carcinoma (20%), breast invasive carcinoma (19%), lung adenocarcinoma (15%), lung squamous cell carcinoma (12%), and kidney renal clear cell carcinoma (6%) (www.cbioportal.org). These findings are consistent with reports of elevated ABL2 expression in advanced high-grade breast, colorectal, pancreatic, renal, and gastric tumors (Nature 2012; Simpson et al., J. Urol., 2005; Behbahani et al., World J. Urology, 2012; Crnogorac-Jurcevic et al., Oncogene, 2002; Wu et al., Anticancer Research, 2003). While ABL2 amplification and increased mRNA levels are genomic alterations found in a subset of human cancers, somatic mutations of ABL1 and ABL2 in solid tumors are rare, but have been reported in lung cancer and uterine corpus endometrioid carcinoma among other cancers (www.cbioportal.org). The role of these mutations in regulating ABL oncogenic activity remains to be determined.
Enhanced activation of the ABL kinases downstream of multiple receptor tyrosine kinases (RTKs), including the PDGFR, the ErbB family member EGF receptor (EGFR), and the hepatocyte growth factor receptor (MET), has been reported by multiple groups (Greuber et al., Nat. Rev. Cancer, 2013; Li et al., PLoS One, 2015; Fiore et al., Oncogene, 2014). Cancer cells expressing activated ErbB receptors exhibited rapid EGF-induced ABL kinase stimulation (Jones et al. Nature, 2006). Subsequent studies demonstrated that ABL kinases are tyrosine phosphorylated and activated in breast, lung, colorectal, gastric, renal, and prostate cancer cells, as well as in melanoma (Greuber et al., Nat. Rev. Cancer, 2013; Ganguly et al., Oncogene, 2012). The catalytic activity of the ABL kinases can be upregulated by ligand-dependent and ligand-independent activation of RTKs in cancer cells. Activation of ABL kinases in breast cancer cells has been reported to occur downstream of the EGFR, Her2 (ERBB2), insulin-like growth factor receptor (IGFR), and the CXCR4 chemokine receptor (Greuber et al., Nat. Rev. Cancer, 2013; Ganguly et al., Oncogene, 2012). ABL1 activation downstream of ligand-activated MET was shown in gastric carcinoma and hepatocellular carcinoma cells (Furlan et al., Cell Death Diff., 2011), and ABL1 activation in human anaplastic thyroid carcinoma cells was induced by a constitutively active form of the receptor tyrosine kinase RET (Iavarone et al., J. Biol. Chem., 2006).
ABL-Dependent Regulation of Cancer Cell Proliferation
The EPHB2 receptor tyrosine kinase can function as an oncogene during adenoma development and as a tumor suppressor in the progression of invasive colorectal cancer (Genander et al., Cell, 2009; Cortina et al., Nature Genetics, 2007). Genetic studies with ABL1-null mice showed that ABL1 is required for EPHB2-mediated proliferation in the small intestine and epithelium because deletion of ABL1 reduced the number of proliferating cells in these tissues (Genander et al., Cell, 2009). Inactivation of ABL1 in the Apcmin/+ mouse model of intestinal adenoma impaired EPHB2-mediated tumor promotion without affecting its tumor suppressor function (Genander et al., Cell, 2009; Kundu et al., Science Trans. Med., 2015). Further, ABL1 inactivation inhibited tumor initiation by intestinal stem cells, decreased tumor load, and extended the lifespan of Apcmin/+ mice (Kundu et al., Science Trans. Med., 2015). Interestingly, ABL1 knockdown or pharmacological inhibition in some human colon carcinoma cell lines expressing low levels of EPHB2 resulted in decreased levels of cyclin D1 and impaired cell proliferation (FIG. 2). Thus, ABL activity and function may become dissociated from EPHB2 signaling at later stages of adenocarcinoma progression.
ABL1 and ABL2 may have distinct roles in the regulation of breast cancer cell proliferation. Pharmacological inhibition or knockdown of ABL1 alone in MDA-MB-231 breast cancer cells and human mammary epithelial cells overexpressing nuclear geminin, a protein implicated in the regulation of chromosomal integrity, markedly decreased the growth of orthotopic mammary tumors (Blanchard et al., PLoS One, 2014). By contrast, knockdown of ABL2 alone in MDA-MB-231 breast cancer cells increased primary tumor size owing to enhanced cell proliferation (Gil-Henn et al., Oncogene, 2012). These results suggest that ABL1 and ABL2 may have opposing effects in the regulation of cell proliferation in some breast tumor types.
ABL-Mediated Metabolism and Oxidative Stress in Cancer
A recent breakthrough study revealed a crucial role for ABL1 in an aggressive form of hereditary kidney cancer (Sourbier et al., Cancer Cell, 2014). Patients with a germline mutation in fumarate hydratase (FH) are susceptible to the development of hereditary leimyomatosis and renal cell carcinoma (HLRCC). FH-deficient renal tumors are highly glycolytic, accumulate high levels of fumarate, lactate, and hypoxia stimulated transcription factor (HIF1α), and have decreased activity of AMP-activated kinase (AMPK) (Yang et al. PLoS One, 2013). The ABL1 kinase was found to be hyperactive in FH-deficient renal cancer cells in response to high fumarate levels (FIG. 2). Mechanistically, activation of ABL1 in HLRCC functions to promote aerobic glycolysis through activation of the mTOR-HIF1α pathway and also induces nuclear localization of the antioxidant response transcription factor NRF2 (FIG. 2). Thus, high ABL1 activity enables these tumors to simultaneously meet their high energetic needs and to neutralize the elevated levels of oxidative stress generated by excess fumarate accumulation in HLRCC. Importantly, ABL1 knockdown or inhibition with either imatinib or vandetanib (an inhibitor that also targets EGFR, RET, and VEGFR; Table 1), was cytotoxic to FH-deficient HLRCC (Sourbier et al., Cancer Cell, 2014). The anti-tumor activity of vandetanib in these cells was shown to be ABL1-dependent. Moreover, vandetanib was shown to potently inhibit the ABL1 kinase (IC50=15 nM) in vitro and in cells. Vandetanib alone markedly inhibited the growth of HLRCC xenografts, and a combination of low-dose vandetanib with the AMPK activator metformin induced complete regression of the HLRCC tumors in 100% of the treated mice (Sourbier et al., Cancer Cell, 2014). ABL kinases have been shown to be activated in response to oxidative stress and reactive oxygen species (ROS) (Sun et al., J. Biol. Chem., 2000). Elevated levels of ROS are a feature characteristic of many solid tumors, and are also an inevitable byproduct of cellular metabolism. Thus, the data on the role for ABL1 in HLRCC suggest that ABL1 kinase inhibitors could be developed for the treatment of FH-deficient tumors and other cancers with high levels of oxidative and metabolic stress.
Role of ABL Kinases in Cancer Cell Invasion and Metastasis
The progression of solid tumors requires invasion of primary tumor cells into the surrounding tissue, followed by intravasation, migration, extravasation, and formation of metastases at distant sites (Fidler, Nat. Rev. Cancer, 2003). The various steps in the metastatic cascade require dynamic remodeling of the actin cytoskeleton. ABL kinases have been shown to engage the actin polymerization machinery to promote formation of membrane protrusions, morphological changes, altered cell adhesion, migration, and invasion of diverse cell types (Bradley et al., J. Cell Sci., 2009). Among the various functions of the ABL kinases, regulation of cell motility has been shown to be a predominant and evolutionarily conserved role for these kinases. A requirement for ABL kinases in cancer cell motility and invasion was shown downstream of IGF-1, EGF, serum, and chemokines (Greuber et al., Nat. Rev. Cancer, 2013). This requirement is consistent with the localization of ABL2 to invadopodia, which are actin-rich, protrusive membrane structures that promote remodeling of the extracellular matrix during tumor invasion (Smith-Pearson et al., J. Biol. Chem., 2010; Mader et al., Cancer Res., 2011). ABL kinases promote maturation of invadopodia and are required for matrix degradation and invasion in some but not all breast cancer types (Smith-Pearson et al., J. Biol. Chem., 2010; Made et al., Cancer Res., 2011; Chevalier et al., PLoS One, 2015). Among the actin cytoskeleton regulatory proteins targeted by ABL kinases at invadopodia are cortactin, N-WASP, WAVE, and the ABL interactor 1 (ABI1) adaptor protein (FIG. 2). Importantly, ABL kinases regulate the expression, localization, and activity of matrix metalloproteinase (MMP) during invadopodia maturation. Active ABL2 interacts with and promotes phosphorylation of the membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), and is required for its localization and function at invadopodia (Smith-Pearson et al., J. Biol. Chem., 2010). Both ABL1 and ABL2 kinases were shown to regulate MMPs expression through STAT3-dependent and -independent pathways in melanoma cells (Ganguly et al., Oncogene, 2012). Knockdown of ABL2 alone decreased cancer cell invasion and intravasation following implantation of MDA-MB-231 cells in the mammary fat pad (Gil-Henn et al., Oncogene, 2012). A requirement for ABL kinases for invasion and metastasis of melanoma cells was also shown, which may be mediated in part by the NM23-H1 metastasis suppressor (Fiore et al., Oncogene, 2014). Active ABL kinases induced cathepsin-dependent lysosomal degradation of NM23-H1 in melanoma and breast cancer cells.
Role of ABL Kinases in Lung Cancer
Lung cancer is the leading cause of cancer mortality worldwide with a five-year survival rate of only ˜10 to 15%, and often results in metastasis to the brain, bone and other organs. Among major drivers of lung cancer are activating mutations in RTKs and KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) as well as loss of tumor suppressors such as TP53, PTEN and LKB1/STK11. Unfortunately, targeted therapies against oncogenic RTKs have shown limited efficacy in the treatment of lung cancer patients due to intrinsic or acquired resistance. Similarly, patients with KRAS-mutant lung cancer exhibit poor outcome and have few tractable therapeutic options.
Role of ABL Kinases in Colorectal Cancer
A recent report demonstrated a novel role for ABL kinases in promoting colorectal cancer cell invasion and metastasis by linking the activation of Notch to the phosphorylation of TRIO (pY2681), leading to enhanced TRIO Rho-GEF activity and a corresponding increase of Rho-GTP levels (Sonoshita et al., Cancer Discov., 2015). Activation of Notch by homozygous deletion of Aes (amino-terminal enhancer of split) in the intestinal epithelium of Apc+/Δ716 polyposis mice resulted in enhanced RBPJ-mediated transcription, leading to increased levels of DAB1, a substrate and activator of the ABL kinases. Activated ABL in colorectal cancer cells induced tyrosine phosphorylation of TRIO on Y2681, leading to enhanced TRIO Rho-GEF activity (FIG. 2). Rho activation in colorectal cancer cells promoted invasion, extravasation and metastasis. Importantly, inhibition of ABL kinases in Apc/Aes compound knockout mice dramatically suppressed both invasion and intravasation incidence without affecting tumor size. These findings suggest that ABL kinases may function to link activation of other cell surface receptors to Rho signaling in different tumors. In this regard, it has also recently been shown that ABL kinases link the ligand-activated MET receptor tyrosine kinase to Rho activation that is required for cell scattering, tubulogenesis, migration, and invasion (Li et al., PLoS One, 2015).
Role of ABL Kinases in Metastatic Breast Cancer
Bone metastases occur in up to 70% of patients with advanced breast cancer and are associated with high mortality and morbidity (Weilbaecher et al., Nat. Rev. Cancer, 2011; Waning et al., Clin. Cancer Res., 2014). Whereas the mechanisms that drive tumor cell homing, invasion, and colonization to the bone are poorly understood, it is increasingly apparent that bone metastasis requires interactions between tumor and stromal cells in the bone microenvironment (Cicek et al., Cancer Metastasis Rev., 2006). For most patients with breast cancer, bone metastases are predominantly osteolytic. When breast cancer cells invade the bone microenvironment, they produce molecules that activate osteoclastic bone resorption, leading to the release of growth factors stored in the bone matrix to promote tumor growth. Currently, there are no available therapies to cure breast cancer metastasis. Thus, there is a need to identify molecules that could be targeted simultaneously in tumor and bone to disrupt the tumor cell-stromal cell interactions that drive metastasis.
Role for ABL Kinases in Cancer Drug Resistance
Enhanced activation of the ABL kinases has been reported in some cancers that have intrinsic or acquired resistance to chemotherapy. Hyper activation of both ABL1 and PDGFR was detected in aromatase inhibitor (AI)-resistant breast cancer patient specimens (Weigel et al., Breast Cancer Res., 2012). ABL1 expression increased at the point of relapse in AI-treated patients, and correlated with increased expression of the Ki67 proliferation marker. In vitro studies showed that estrogen deprivation of MCF7 breast cancer cells, which became AI-resistant, was accompanied by up-regulation of PDGFR and ABL1 signaling (Weigel et al., Breast Cancer Res., 2012). Treatment of these cells with nilotinib, a PDGFR and ABL inhibitor, suppressed proliferation and estrogen receptor (ER)-mediated transcription, in part by destabilizing the ER protein. Down regulation of ABL1 in some human breast cancer cell lines by RNA interference or imatinib treatment was reported to overcome resistance to fulvestrant, a compound that down regulates ERα levels and activity (Zhao et al., Mol. Carcinogenesis, 2011). Furthermore, in vitro studies using breast cancer cells resistant to lapatinib, an EGFR and ErbB2 inhibitor, showed that imatinib treatment or ABL1 depletion restored lapatinib sensitivity to these breast cancer cells (Lo et al., Anticancer Research, 2011). These studies suggest that inhibition of the ABL kinases may be effective in overcoming cancer cell resistance to diverse therapeutic agents.
A role for ABL kinase inhibitors in reversing resistance to doxorubicin in breast cancer (BT-549 and MDA-MB-468) and melanoma (WM3248) cell lines has been linked to at least two pathways (Sims et al., PLoS One, 2013). Imatinib blocked intrinsic resistance to doxorubicin by inhibiting STAT3-mediated cell survival and repressing NF-kB target gene expression. In addition, imatinib prevented acquired resistance by inhibiting the increased expression of the ABCB1 drug transporter, which mediates efflux of chemotherapeutic compounds such as doxorubicin. Similar to imatinib, other ATP-competitive inhibitors (nilotinib and dasatinib) have been reported to sensitize cancer cells to cytotoxic chemotherapies and targeted TKI therapies. However, the majority of these studies was carried out with ABL TKIs, and did not evaluate whether these effects were mediated specifically by inactivation of the ABL1 and/or ABL2 kinases in the cancer cells or in associated cells in the tumor microenvironment.
Targeting ABL Kinases in Endothelial Cells and Fibroblasts
Endothelial cells (ECs) and cancer-associated fibroblasts contribute to tumor progression and metastasis. TKIs such as imatinib have anti-angiogenic activity. For example, imatinib treatment of a mouse model of cervical cancer impaired angiogenesis in part by blocking the function of cancer-associated fibroblasts (Raimondi et al., J. Ex. Med., 2014). The anti-angiogenic effects of imatinib have been largely attributed to inhibition of the PDGFR. However, ABL kinases, which are also targeted by imatinib, regulate diverse cellular processes in both ECs and fibroblasts. Conditional deletion of ABL1 in ECs in ABL2-null mice resulted in late-stage embryonic and perinatal lethality (Chislock et al., Proc. Natl. Acad. Sci. USA, 2013). Loss of ABL kinases led to increased endothelial cell apoptosis. ABL kinases play a dual role in angiopoietin (Angpt)/Tie2 signaling by regulating both Tie2 expression and activation of Tie2-mediated pathways required for cell survival. ABL kinases are also required for induction of endothelial permeability by VEGF and other factors (Sirvent et al., Biology of the Cell, 2008). Inactivation of the ABL kinases with pharmacological inhibitors or genetic inactivation in mice impaired VEGF-induced vascular permeability. Recently, ABL1 was shown to interact with neuropolin (NRP1) in human dermal microvascular ECs and link fibronectin-dependent activation of NRP1 to paxilin phosphorylation, actin remodeling, and EC mobility (Bi et al., Am. J. Path., 2014). Moreover, ABL kinases regulate signaling downstream of multiple cell surface receptors in fibroblasts. ABL kinases are activated by ligand-activated PDGF receptor, leading to fibroblast proliferation and mobility (Yaqoob et al., Cancer Res., 2012). ABL1 can also be activated downstream of the lipid second-messenger sphingosine 1 phosphate (S1P) and its receptor, leading to RAC activation and cytoskeletal remodeling required for fibroblast migration and invasion. ABL1 promotes S1P-dependent reciprocal signaling between stellateate cells and pancreatic cancer cells that is required for NF-κB activation and MMP9 production (Sun et al., Carcinogenesis, 2009). ABL1 also functions downstream of NRP1 in stromal myofibroblasts to induce integrin activation and fibronectin fibril assembly in the tumor microenvironment (Srinivasan et al., Cancer Res., 2006). Thus, pharmacological inhibitors target ABL signaling not only the in tumor cells but also in the various cell types populating the tumor stroma, including ECs and fibroblasts, and may function to blunt angiogenesis through multiple pathways.
While imatinib sensitizes some breast cancer cells to apoptosis by treatment with cisplatin and other chemotherapeutic agents (Sims et al., Biochem. Pharm., 2009), imatinib or GNF2 treatment was reported to protect mouse oocytes against cisplatin-induced cell death (Gonfloni et al., Nat. Med., 2009). The disparate responses by germ cells versus cancer cells to DNA-damaging agents in the presence of ABL kinase inhibitors may be due to differential roles for ABL1 in the regulation of double-strand breaks and DNA damage signaling (Gonfloni, Oncogene, 2010). Further, different cellular responses may be elicited depending on the status of TP53 or its homolog TAp63, ABL1 enzymatic activity levels, ABL1 nuclear versus cytoplasmic localization, and the extent of DNA damage.
Therapeutic Potential for Tyrosine Kinase Inhibitors in Solid Tumors
The development of TKIs to treat patients with BCR-ABL1-positive leukemias is the best example of the successful application of targeted therapy. In contrast to the success of ATP-competitive inhibitors imatinib, nilotinib, and dasatinib in treating BCR-ABL1-induced leukemias, treatment of diverse solid tumors with these compounds has not achieved similar success (Ganguly et al., Genes & Cancer, 2012; Puls et al., The Oncologist, 2011). Drugs such as imatinib and nilotinib, shown to inhibit ABL kinases, have demonstrated mixed effectiveness for the treatment of solid tumors. The variable clinical responses to these TKIs may be due to the lack of the relevant oncogenic target, the presence of additional mutations driving the tumor, tumor heterogeneity, and/or dynamic reprogramming of signaling networks in response to TKI treatment (Stuhlmiller, et al., Cell Rep., 2014; Duncan et al., Cell, 2012).
An alternative mechanism that underlies the poor response to TKI therapy is the paradoxical activation of proliferative pathways as a result of unintended targeting of other kinases. Imatinib, dasatinib and nilotinib, which have multiple cellular targets, drive the paradoxical activation of BRAF/C-RAF complexes leading to enhanced activation of the MEK-ERK pathway. This was demonstrated by the activation of BRAF/RAF1 complexes leading to enhanced activation of the MEK-ERK pathway by nilotinib, imatinib, and dasatinib in melanoma, lung, colorectal, pancreatic carcinoma cells, and BCR-ABL1 TKI-resistant leukemic cells expressing activated RAS (Packer et al., Cancer Cell, 2011). It is clear that the use ATP-competitive inhibitor drugs is inadequate for the treatment of solid tumors as monotherapies owing to the complexity of mutations even in early-stage tumors, and the potential for inappropriate activation (rather than inhibition) of proliferative pathways by some TKIs with multiple protein targets. In contrast to the ABL-targeted ATP-competitive TKIs, the ABL allosteric inhibitors GNF2 and GNF5, targeting the unique myristate binding site in the ABL kinase domain, do not induce the formation of BRAF/RAF1 dimers, and fail to elicit paradoxical activation of RAF-ERK signaling (Packer et al., Cancer Cell, 2011; and FIG. 14, respectively, for GNF2 and GNF5). To date no studies appear to have directly evaluated the consequences of specifically targeting the ABL kinases with selective kinase inhibitors in solid tumors including breast cancer.
Cancer cell types with hyper-activation of the ABL kinases as a consequence of amplification, enhanced expression, and/or elevated kinase activity would be more likely to rely on ABL signaling for tumor progression and metastasis. Thus, these cancer subtypes might benefit from treatment with ABL-selective TKIs such as the new allosteric inhibitors, resulting in the inhibition of ABL-dependent pathways in the tumor and associated stromal cells including endothelial cells, fibroblasts, and infiltrating myeloid cells.
The use of specific ABL-dependent signatures (genomic, transcriptional, or phospho-proteomic) in various tumors and associated stroma may be useful for the identification of those solid tumor types that might benefit from the use of ABL TKIs, in combination with other agents, to impair metastatic progression and block the development of chemo-resistance. Thus, it may be important to identify those tumors that may benefit from therapies with selective ABL TKIs in combinations to prevent the emergence of therapy resistance.